P1349: EXTRACELLULAR ATP CONTRIBUTES TO ERYTHROPOIESIS-SUPPORTIVE MICROENVIRONMENT UNDER CHRONIC STRESS CONDITIONS
Authors: Sanja Momčilović, Andrija Bogdanovic, Maja Milošević, Danijel Jakovljević, Aleksandra Jauković, Diana Bugarski, Sanja Vignjević Petrinović
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Extracellular adenosine triphosphate (ATP) acts as a signal of disturbed tissue homeostasis and triggers activation of purinergic receptors, thereby affecting various cellular processes such as proliferation, differentiation, and migration. Macrophages migrate toward increasing ATP concentrations and possess P2X7 receptors (P2X7R), which respond to high ATP levels, as well as the ectonucleotidase CD39 that rapidly convert ATP to adenosine monophosphate. Using a mouse model of psychological stress, we recently demonstrated that macrophages substantially support erythropoiesis under chronic stress conditions, but the role of extracellular ATP in stress-induced erythropoiesis remains unknown. Aims: This study was undertaken to investigate the effects of chronic stress on i) extracellular ATP levels, ii) P2X7R expression, and iii) CD39 activity and expression in mouse bone marrow and spleen, and the role of macrophages in stress-induced changes. Methods: Adult male BALB/c mice were subjected to 2h daily restraint stress for 7 consecutive days. Clodronate liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to first restraint procedure, as well as newly recruited macrophages every third day for the duration of the experiment. Single-cell suspensions of bone marrow and spleen samples were obtained from each mouse and 1×106 cells were plated in duplicate. Following the incubation time of 4 h, cell culture supernatants were collected for quantification of extracellular ATP concentrations by the ATP bioluminescence assay. Bone marrow and splenic CD39 cell activity was measured using the malachite green phosphate assay, while the expression levels of CD39 and P2X7R genes in the bone marrow and spleen were determined by quantitative real-time PCR analysis. Results: Chronic exposure to stress markedly increased the extracellular ATP levels in both bone marrow and spleen. Macrophage depletion significantly reduced this stress-induced increase in bone marrow and splenic extracellular ATP concentrations. Changes in extracellular ATP levels among different experimental groups closely resemble those in the number of bone marrow and splenic erythroid progenitor cells previously observed among the same groups (Momčilovic et al. HemaSphere 2022; 6:2469). The increased activity of CD39 was detected in bone marrow and spleen of chronically stressed mice, whereas macrophage depletion abolished this effect of stress. The expression of CD39 and P2X7R were significantly upregulated in the bone marrow and spleen of chronically stressed mice. Treatment with clodronate liposomes did not alter CD39 and P2X7R expression levels under basal conditions but prevented stress-induced elevation of CD39 and P2X7R expression in the bone marrow and spleen. Summary/Conclusion: The obtained results demonstrate a stress-induced increase in extracellular ATP levels, P2X7R expression, and in both CD39 activity and expression within bone marrow and spleen, as well as a significant role of macrophages in stress-induced changes. These findings together with a close resemblance between the stress-induced changes in erythroid progenitor cell number and in extracellular ATP levels suggest that extracellular ATP contributes to erythropoiesis-supportive microenvironment under chronic stress conditions. Keywords: Erythropoieisis, Macrophage